1. Field of the Invention
The present invention relates to a novel process for dehydrogenation of azaandrostane compounds.
2. Related Art
The 4-azasteroids are well known specific inhibitors of the testosterone 5xcex1-reductase enzyme. According to previously published data it is apparent that the introduction of the double bond in the C-1,2 position of the molecule is necessary for obtaining the desired effect.
There are both synthetic and biochemical methods for introducing a double bond into the C-1,2 position of 4-azasteroids.
One of the synthetic methods is the introduction of the xcex941 double bond with benzeneselenic anhydride in boiling chlorobenzene (for example, Patent No. EP 155 096).
Alternatively, dehydrogenation can be carried out with kinones (e.g. 2,3-dichloro-5,6-dicyano-benzokinone) in the presence of silylating agents (e.g., bis-trimethyl-silyl-trifluoro-acetamide) (Patent No. EP 298 652).
According to processes described in EP 428 366 and EP 655 459 the 2-halogen derivative of a steroid can be used as an intermediate to introduce the C-1,2 double bond. Patent No. CA 2 271 974 discloses a process in which the xcex941 double bond is introduced into an aza-steroid using a 2,2-dibromo-aza-steroid compound as an intermediate.
In another process, 2-halogen derivatives are prepared from the appropriate unsubstituted 4-aza compound via a trialkylsilyl-trifluoro-methanesulfonate intermediate with iodine or trimethylsilyl chloride and iodine (Patent No. EP 473 226). The desired xcex941 compound is obtained from this 2-halogen-4-aza derivative with potassium tert-butoxide in dimethylformamide solution in about 60% yield.
These synthetic methods require extremely aggressive reaction conditions that have several disadvantages. First, impurities are formed. Second, the impurities, although formed in low concentration (below 0.1%), are not characterized, and some might be toxic. In addition, some of the applied reagents are carcinogenic, flammable, demand completely dry circumstances, or are highly corrosive, and therefore are environmental hazards.
In contrast, the conditions required for biochemical methods for xcex941 dehydrogenation are gentle and produce fewer and less environmentally hazardous by-products. As a consequence, the formation of toxic compounds is less likely. Moreover, instead of two synthetic steps, the transformation can be carried out in a single step.
Although the xcex941-dehydrogenation of steroids is well known in the literature (Microbial Transformations of Steroids, Charney, W., Herzog, H. L., Academic Press, New York, London, (1967)), there are very few examples of the microbiological introduction of a double bond into the C-1,2 position of aza-steroids, probably because of the well known antibacterial effect of aza-steroids (J. Bacteriol., 93(2), 627-35 (1967); Steroids, 27(4), 525-41 (1976); Chem. Ind., 52, 1660-1 (1970)).
The microbial introduction of a xcex941 double bond into 12a-aza compounds has been accomplished with Nocardia sp. and Arthrobacter sp. strains (Mazur et al., J. Org. Chem., 28(9), 2442-3 (1963)). According to Mazur et al., the conversion of 12a-aza-C-homo-1,4-pregnadiene-3,12,20-trione could be realized at 0.29 g/L concentration with a 62% yield; the conversion of 12a-aza- 17xcex1-hydroxy-C-homo-1,4-pregnadiene-3,12,20-trione could be realized at 0.17 g/L concentration with a 17% yield; and the conversion of 12a-aza-C-homo-5xcex1-pregn-1-ene-3,12,20-trione could be realized at 0.29 g/L concentration with a 26% yield.
Similarly, Arthrobacter sp. and Nocardia sp. strains have been used to xcex941-dehydrogenate the 17-N-tert-butylcarbamoyl-4-azaandrostan-3-one substrate at 0.1-1.0 g/L concentration with a 20-80% yield (Patent No. EP 786 011). In this method, the xcex941-dehydrogenase enzymes of the above strains were induced by the addition of hydrocortisone, the separated biomass was suspended in a buffer (pH=6-8) saturated with organic solvent and the bioconversion was performed in the presence of menadione or phenazine methosulfate. The bioconversion was carried out in a small volume. In the only disclosed example the conversion was carried out for 3 days in a volume of 20 ml at 0.26 g/L substrate concentration with a 60% yield. However, for industrial realization, one must determine the appropriate concentration level and scale up the procedure. According to our experiments, when this procedure is scaled up, the conversion drops drastically. Therefore, a fundamental modification of this process is essential for industrial realization.
The aim of the present invention is to provide a bioconversion process that avoids the disadvantages of wholly synthetic methods while achieving industrial realization. More specifically, the invention relates to a process for the production of compounds of general formula (I), wherein R1 is a xe2x80x94NH-tert-butyl group or a 4-methyl-piperidino group, by bioconversion of compounds of general formula (II), wherein R1 is as described above, using a biocatalyst having steroid-xcex941-dehydrogenase enzyme activity, wherein the activity of the enzyme needed for the bioconversion is produced by induction. 
In one preferred embodiment, the present invention encompasses a method for producing a compound of formula I, comprising the steps of:
a) preparing a culture of a biocatalyst or an extract of a biocatalyst;
b) adding to the culture or extract an inducer of steroid-xcex941-dehydrogenase activity;
c) adding to the culture or extract an electron carrier, a stabilizer, and a compound of formula II; and
d) incubating the culture or extract for a time sufficient for bioconversion of the compound of formula II to the compound of formula I to occur.
In another preferred embodiment, the present invention encompasses a method for producing a compound of formula I, comprising the steps of:
a) preparing a culture of a biocatalyst or an extract of a biocatalyst, wherein said culture or extract has a volume of at least about 1 L;
b) adding to the culture or extract an inducer of steroid-xcex941-dehydrogenase;
c) adding to the culture or extract an electron carrier, a stabilizer, a complexing agent or emulsifier, and a compound of formula II; and
d) incubating the culture or extract for a time sufficient for bioconversion of the compound of formula II to the compound of formula I to occur.
In another preferred embodiment, the present invention encompasses a method for producing a compound of formula I, comprising the steps of:
a) preparing a culture of a biocatalyst or an extract of a biocatalyst;
b) adding to the culture or extract an inducer of steroid-xcex941-dehydrogenase;
c) adding to the culture or extract an electron carrier at a concentration of about 0.05 to about 3.5 g/L and a stabilizer at a concentration of about 0.01 to about 0.1 g/L and a compound of formula II; and
d) incubating the culture or extract for a time sufficient for bioconversion of the compound of formula II to the compound of formula I to occur; provided that an oxygen scavenger is not added to the culture or extract.
The invention relates to a process for the production of compounds of general formula (I), wherein R1 is a xe2x80x94NH-tert-butyl group or a 4-methyl-piperidino group, by bioconversion of compounds of general formula (II), wherein R1 is as described above, using a biocatalyst having steroid-xcex941-dehydrogenase enzyme activity, by inducing the activity of the enzyme, maintaining the activity of the enzyme at the necessary level by adding a stabilizer, promoting the continuous dissolution of the substrate by adding a complexing agent, and completing the conversion by adding an electron carrier.
The term bioconversion is defined as the conversion of compound (II) to compound (I) through the use of a biocatalyst such that at least a measurable amount of compound (I) is produced.
The term biocatalyst is defined as any microorganism that contains or can be induced to contain steroid-xcex941-dehydrogenase enzyme activity. Examples of biocatalysts include, but are not limited to, bacteria, fungi and animal cells. The method of the invention may be carried out with the above mentioned and known methods of preparing a biocatalyst (e.g., intact cells, cell free extracts, immobilized preparations thereof). For example, intact cells of Arthrobacter simplex can be used by incubating a submerged culture in a liquid medium containing a carbon source, preferably glucose in a concentration of 1-15 g/L, a nitrogen source, such as yeast extract in a concentration of 1-10 g/L, and inorganic salts. The culture is incubated at 25-38xc2x0 C., preferably at 35xc2x0 C., for 20-72 hours. A solution containing the agent(s) inducing the activity of the steroid xcex941-dehydrogenase enzyme is then added. For those skilled in the art, it will be apparent that the process of the invention can be carried out with other biocatalysts having steroid-xcex941-dehydrogenase activity, such as living or resting intact cells, cell lysate and free or immobilized preparations (J. Am. Chem. Soc., 108, 6732, (1986); Chem. Rev., 92, 1071-1140 (1992)).
The strains Arthrobacter sp. and Nocardia sp. may be used in the methods of the invention. Moreover, although azasteroids have an antibacterial effect, surprisingly we found that, in addition to the Arthrobacter sp. and Nocardia sp., strains belonging to the genera Bacillus and Mycobacterium were able to xcex941-dehydrogenate 4-azasteroids. Specific strains that may be used include, but are not limited to, Arthrobacter simplex (ATCC 6946), Bacillus subtilis (NRRL B-558), Bacillus sphaericus (ATCC 7054), Bacillus lentus (ATCC 13805), Nocardia corallina (ATCC 4275), and Mycobacterium sp. (NRRL B-3683). The production of cell free extracts of steroid-xcex941-dehydrogenase, as well as the immobilization of intact cells or cell free extracts from all of these strains is widely described in the literature (J. Am. Chem. Soc., 108, 6732, (1986); Chem. Rev., 92, 1071-1140 (1992)).
The activity of the xcex941-dehydrogenase enzyme is induced by a known method, such as the addition of hydrocortisone. Other inducers that may be used include, but are not limited to, prednisolone, prednisone, cortisone, and androsta-1,4-diene-3,17-dione. However, the activity of the enzyme decreases over the course of time due to the consumption of the coenzyme or the activity of proteolytic enzymes. Several known methods may be used to maintain the activity of the enzyme; such as chemical modification or immobilization of the enzyme, packing the enzyme into a liposome capsule, and the use of additives (sugar derivatives: polyols, esters; amino acids, proteins, dextrins, lecitin, DMSO). World Biotech. Rep., 1, 379-390 (1984); Patent No. JP 62-096084; Biotechnol. Bioengineer. 75, 615-618 (2001); Patent Nos. JP 96196330, JP 89185636, JP 86253336.
In the absence of an induction agent, the activity of the xcex941-dehydrogenase enzyme may drop. To compensate for this decrease, it is necessary to add more of the induction agent. For a bioconversion that continues for several days the induction agent must be added continuously in order to maintain the necessary concentration of induction agent.
The induction of the enzyme may be performed by any known method, such as by addition of a methanolic solution of hydrocortisone into the culture in a concentration of about 10 to about 100 mg/L. Since hydrocortisone is enzymatically degraded, its induction effect decreases continuously. Therefore, to maintain the high enzyme activity necessary for bioconversion, repeated addition of hydrocortisone (altogether 2.7 g/L) is required. The frequency of hydrocortisone administration is dependent on the rate of enzymatic breakdown of the hydrocortisone. Preferably, hydrocortisone is administered once per day.
According to a more preferred method, the induction can be performed by addition of steroids that are resistant to degradation by bacterial enzymes, such as 9xcex1-hydroxylase, and that are substrates of the xcex941-dehydrogenase enzyme, or that already have a xcex941-double bond, in a concentration of about 10 to about 100 mg/L.
Surprisingly, we have had excellent results with the bioconversion process by, after the induction of the activity of the xcex941-dehydrogenase enzyme, adding a stabilizer to maintain the appropriate level of enzyme, adding a complexing agent to promote continuous dissolution of the substrate, and adding a known electron carrier to make the bioconversion complete.
It was found that compounds that cannot induce the activity of xcex941-dehydrogenase enzyme can stabilize the enzymes already present in the system and preserve their xcex941-dehydrogenase activity for a prolonged time without any other intervention. 5xcex1-androstanediol or its 17-methyl derivative, 13-ethyl-gon-4-ene-3,17-dione or its acetoxy or hydroxy derivative, 9,11-dehydro-hydrocortisone or its acetate derivative, estr-4-ene-3,17-dione or its acetoxy or 11xcex1-hydroxy derivative, and 13-ethyl-10,11xcex1-dihydroxy-gon-4-ene-3,17-dione, which are added to stabilize the enzyme, can preserve the activity when added in a final concentration of about 10 to about 100 mg/L. The most preferred stabilizer is 13-ethyl-10,11xcex1-dihydroxy-gon-4-ene-3,17-dione, which maintains the induction effect at 50 mg/L concentration for days with no loss of dehydrogenase activity experienced for 3-5 weeks when the culture is stored at 5-12xc2x0 C.
The vast majority of steroids are poorly soluble in water. However, the addition of a steroid substrate dissolved in an organic solvent would not provide sufficient bioavailability in the bioconversion system. Thus, emulsifiers or complexing agents may be used in the method of the present invention to increase biological accessibility of the substrate in bioconversions performed in aqueous systems. An emulsifier, such as polyoxyethylene-sorbitan-monooleate (e.g., TWEEN-80) (Patent No. HU 216 874) in a concentration of about 0.5 to about 4 g/L may be used. Other emulsifiers that may be used include, but are not limited to, TEGIN(copyright) and TAGAT(copyright) (Goldschmidt Chemical Corp.). Alternatively, a complexing agent, for example, xcex1-, xcex2- and xcex3-cyclodextrins and cyclodextrin derivatives, may be used advantageously ( Appl. Microbiol. Biotechnol., 32, 556-559 (1990); xe2x80x9cCyclodextrins and their Industrial Usesxe2x80x9d ed. D. Duchene, Editions de Sante, Paris, (1987); Appl. Microbiol. Biotechnol., 58, 393-399 (2002); Patent No. RU 2 039 824). Another complexing agent that may be used is TAMOL(copyright) (BASF).
In the process of the present invention, cyclodextrin and cyclodextrin derivatives may be added to the medium in a concentration of about 1 to about 50 g/L. Methyl-xcex2-cyclodextrin and hydroxypropyl-xcex2-cyclodextrin are preferred. A cyclodextrin derivative is defined as a cyclodextrin modified by one or more moieties such that the modified cyclodextrin maintains the ability to act as a complexing agent. Moieties that may be used include methyl, dimethyl, random-methyl, carboxy-methyl, and succinyl.
Since the enzymatic conversion of the substrate of general formula (II) to the product of general formula (I) is an equilibrium reaction, to accomplish the conversion and to increase the reaction rate, according to the process of the invention, electron carriers are used, such as naphthoquinone, menadione, menadione bisulfite and phenazine methosulfate. Preferably, menadione bisulfite is used in a concentration of about 0.05 to about 3.5 g/L, more preferably in a concentration of at least 0.1, 0.5, 1.0, 1.5, or 2.0 g/L, even more preferably in a concentration of about 3.1 g/L. The addition can be performed in one batch or in several portions, but is preferably added in four portions. Preferably, the electron carrier is added at intervals of 18-24 hours.
Note that this element of the process of the invention is quite surprising, since by increasing the amount of the applied electron carrier by two orders of magnitude over the usual amount, the conversion rate can be increased substantially, without using further additives to remove the formed toxic oxygen species. The published preconception was that the use of larger amounts of electron carrier would cause the formation of toxic oxygen derivatives that would have to be removed by oxygen scavengers. See the process described in U.S. Pat. No. 4,749,649.
Applying any one of the three elements of this invention (stabilizer, complexing agent, electron carrier) individually results in a 10-15% increase in the bioconversion over that seen in its absence. However, the combined use of all of them surprisingly gives not a simple additive effect but a synergic effect resulting in a bioconversion of more than 90%.
While the bioconversion can be carried out in any volume, the present invention advantageously is carried out in a volume of at least 1 L, preferably at least 5 L.
The bioconversion can be carried out for a length of time sufficient for at least a measurable amount of bioconversion to occur, preferably at least 24 hours, more preferably at least 72 hours.
The activity of the induced culture can be increased by known methods, for example, by producing a high cell concentration. This can be accomplished by nutrient feeding, repeated inoculation or separation of the cells and resuspending them in a reduced volume. The cells can be resuspended in a buffer solution of pH 6-8, in a buffer solution saturated with organic solvent, or in fresh medium. According to a preferred method, the cells are separated and resuspended in a fresh medium containing the above mentioned ingredients so as to reach five to ten fold concentration. The substrate of general formula (II) is added to this culture of high activity in an ethanolic solution, in about 0.2 to about 2 g/L concentration, preferably in about 1 g/L final concentration.
The following examples are illustrative, but not limiting, of the methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in synthetic chemistry and bioconversion and which are obvious to those skilled in the art are within the spirit and scope of the invention.
The active ingredient content of the crystalline products obtained in the following examples was determined by HPLC.
The conditions of the measurement were as follows:
Column: LiChroCART 250-4, LiChrospher 100 CN (5 xcexcm)
Eluent: tetrahydrofuran : water=30:60
Flow speed: 0.7 ml/min
Injected volume: 20 xcexcL
Detection: UV, 210 nm
Temperature: 60xc2x0 C.